Tissue factor, also called thromboplastin, is a membrane-associated glycoprotein which functions by forming a complex with blood coagulation Factors VII and VIIa. The activity of tissue factor depends on the presence of phospholipids, which are associated with the protein. Bach, Ronald R., "Initiation of Coagulation by Tissue Factor", CRC Critical Reviews in Biochemistry 23(4):339-368 (1988). The complexing of Factors VII and VIIa with tissue factor greatly enhances their proteolytic activity. The complex activates a series of enzymes that comprise the extrinsic and common pathways of the coagulation cascades, ultimately leading to the formation of thrombin, which catalyzes the conversion of fibrinogen into fibrin, resulting in clot formation. Nemerson, Yale, "Tissue Factor and Hemostasis" Blood 71:1-8 (1988).
Diagnostic tests, such as the prothrombin time (PT) test, utilize this series of enzymatic events in vitro under controlled conditions to diagnose deficiencies in the blood coagulation systems of patients, and to monitor patients on anticoagulant therapy. In the PT test, a reagent which induces coagulation is added to a sample of the patient's plasma. All PT reagents contain tissue factor as a coagulation inducing ingredient. The time it takes for clot formation to occur in the plasma sample is the prothrombin time or PT value. Most commercially available PT reagents contain crude tissue factor extracted from natural sources, e.g., rabbit brain, rabbit brain/lung mixtures, human placenta or bovine brain. The crude extracts, because they are natural products, often lack lot to lot uniformity. For example, rabbit brain thromboplastins show seasonal variability. Another problem with natural extracts is the presence of contaminants. For example, human tissue factor may be a source of HIV or other human viral diseases and many natural-sourced thromboplastins also contain other extraneous coagulation factors which can detrimentally affect the PT value.
In an attempt to overcome some of these problems, recombinant tissue factors have been used to make PT reagents. For example, a prothrombin time reagent based on recombinant human tissue factor is described in published PCT Application WO93/07492. However, human tissue factor is sensitive to proteins induced by vitamin K absence or antagonists (PIVKAs), which has been shown to alter the PT time and give an inaccurate result in the PT test (Kovacs M. J. et al, "Assessment of the validity of the INR system for patients with liver impairment." Thromb Haemost, (1994); 71:727-30 and Spaethe, R. and Shirley, I. "Comparison of a highly sensitive rabbit brain thromboplastin, Dade Thromboplastin FS, with a human brain thromboplastin, Manchester Comparative Thromboplastin" in Thromboplastin Calibration and Oral Anticoagulant Control, A.M.H.P. van den Besselaar, H. R. Gralnick and S. M. Lewis, editors. Martinus Nijhoff Publishers, (1984), pp. 197-206). PIVKAs often are present in patients on anticoagulant therapy, or patients having a liver disease or other disorder which causes a vitamin K deficiency. Therefore, PT reagents based on human tissue factor may provide inaccurate PTs when used with plasma samples from patients with elevated PIVKA levels.